JP2006503879A - Method for obtaining a cell line in a protein-free medium and the cell line thus obtained - Google Patents

Method for obtaining a cell line in a protein-free medium and the cell line thus obtained Download PDF

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JP2006503879A
JP2006503879A JP2004545689A JP2004545689A JP2006503879A JP 2006503879 A JP2006503879 A JP 2006503879A JP 2004545689 A JP2004545689 A JP 2004545689A JP 2004545689 A JP2004545689 A JP 2004545689A JP 2006503879 A JP2006503879 A JP 2006503879A
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ペレス・ロドリゲス，ロランド
カステイロ・ビトロシユ，アドルフ
ビトレス・サラゾラ，スビエタ
ボジヤノ・アヨ，タミー
ロハス・デル・カルボ，ルイス
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Abstract

本発明は血清および蛋白質非含有培地中で増殖するように適応される哺乳動物細胞を得る方法に関する。本発明の方法はその間に細胞が蛋白質非含有培地中で生存する能力を獲得しなければならない臨界蛋白質濃度範囲を含んで成る２適応段階から成る方法を含む。各細胞株に対し特定の臨界蛋白質濃度範囲が存在する。本発明は更に、少なくとも４０世代にわたり血清および蛋白質非含有培地中で安定な方法で増殖する哺乳動物の細胞株に関する。本発明は更に、抗−ＣＤ６Ｔ１ｈＴおよびＥＧＦｈＲ３抗受容体ヒト化抗体および抗−ＣＤ３Ｔ３Ｑキメラ抗体並びに前記抗体の断片を発現するクローンに関する。The present invention relates to a method for obtaining mammalian cells adapted to grow in serum and protein free media. The method of the present invention includes a method consisting of two adaptation steps comprising a critical protein concentration range during which the cells must acquire the ability to survive in protein-free medium. There is a specific critical protein concentration range for each cell line. The invention further relates to mammalian cell lines that grow in a stable manner in serum and protein free media for at least 40 generations. The invention further relates to anti-CD6T1hT and EGFhR 3 anti-receptor humanized antibodies and anti-CD 3 T 3 Q chimeric antibodies and clones expressing fragments of said antibodies.

Description

本発明はバイオテクノロジー、特に２段階適応方法による、血清−および蛋白質−非含有培地に適応された安定な細胞クローンを回収する方法に関する。   The present invention relates to a method for recovering stable cell clones adapted to serum- and protein-free media by biotechnology, in particular a two-step adaptation method.

哺乳動物細胞のインビトロの培養の開発以来、それらがもたらす多数の製品の診断的および治療的可能性のために、これらの細胞の大規模な生産の需要が増加した。これらの有用な物質にはモノクロナール抗体、ヒト成長ホルモン、リンホカイン、エリスロポイエチン、血液凝固因子および組織プラスミノーゲン活性化物質が含まれる。   Since the development of in vitro cultures of mammalian cells, the demand for large-scale production of these cells has increased due to the diagnostic and therapeutic potential of the numerous products they provide. These useful substances include monoclonal antibodies, human growth hormone, lymphokine, erythropoietin, blood clotting factor and tissue plasminogen activator.

異なる疾患の治療およびインビボ診断のための組換えモノクロナール抗体（ｒＭａｂ）の使用は多数の場合、高用量の処置の使用を意味する。この事実が非常に高い純度をもつ大量の問題のｒＭａｂの生産を必要にさせる。   The use of recombinant monoclonal antibodies (rMab) for the treatment of different diseases and in vivo diagnosis often means the use of high dose treatments. This fact necessitates the production of large quantities of problematic rMabs with very high purity.

癌および自己免疫疾患の治療および診断における可能な使用を伴う幾つかの組換えモノクロナール抗体が分子免疫学センター（Ｃｅｎｔｅｒ ｏｆ Ｍｏｌｅｃｕｌａｒ Ｉｍｍｕｎｏｌｏｇｙ）においてＮＳＯ骨髄腫細胞中に発現されてきた。特許文献１は前記受容体を発現する腫瘍の診断および治療に有用な表皮成長因子受容体（ＥＧＦ−Ｒ）に対するキメラおよびヒト化抗体の獲得につき記載している（特許文献１参照）。特許文献２は乾癬を罹患する患者の診断および治療に有用な抗−ＣＤ６モノクロナール抗体につき記載している（特許文献２参照）。Ｇａｖｉｌｏｎｄｏ等はＩＯＲ−Ｔ３ａと呼ばれる抗−ＣＤ３モノクロナール抗体を報告した（非特許文献１参照）。   Several recombinant monoclonal antibodies with possible use in the treatment and diagnosis of cancer and autoimmune diseases have been expressed in NSO myeloma cells at the Center of Molecular Immunology. Patent Document 1 describes the acquisition of chimeric and humanized antibodies against epidermal growth factor receptor (EGF-R) useful for diagnosis and treatment of tumors expressing the receptor (see Patent Document 1). Patent Document 2 describes an anti-CD6 monoclonal antibody useful for diagnosis and treatment of patients suffering from psoriasis (see Patent Document 2). Gavilondo et al. Reported an anti-CD3 monoclonal antibody called IOR-T3a (see Non-Patent Document 1).

蛋白質非含有培養状態については、様々な技術が開発されてきた。従って、蛋白質非含有条件下で細胞増殖を可能にする、具体的に定義された完全に蛋白質非含有培地が開発されてきた。   Various techniques have been developed for protein-free culture conditions. Thus, specifically defined completely protein-free media have been developed that allow cell growth under protein-free conditions.

特許文献３は蛋白質非含有条件下における組換え蛋白質の発現につき記載している（特許文献３参照）。   Patent Document 3 describes the expression of a recombinant protein under protein-free conditions (see Patent Document 3).

特許文献４は宿主細胞の産生能力を増加するために非イオン性界面活性剤もしくはシクロデキストリンを添加することによるＣＨＯ細胞中の第ＶＩＩＩ因子の産生のための蛋白質非含有培地の使用につき記載している（特許文献４参照）。これらの添加剤の効果を増強するために、例えばブチレートおよびリチウムの添加が推奨されている。   U.S. Patent No. 6,057,031 describes the use of a protein-free medium for the production of factor VIII in CHO cells by adding a nonionic surfactant or cyclodextrin to increase the production capacity of the host cell. (See Patent Document 4). In order to enhance the effect of these additives, for example, addition of butyrate and lithium is recommended.

特許文献５はその遊離アミノ酸含量が蛋白質の総重量の１５％未満であり、そのペプチドが４４ｋＤ未満の分子量を有するグルタミン含有蛋白質加水分解物を含有する培地中での細胞培養につき記載している（特許文献５参照）。細胞培養のための培地としては、それにはとりわけ、胎児コウシ血清、ゲンタマイシンおよびメルカプト−エタノールが蛋白質加水分解物に加えて添加されている基礎培地として、合成の最少の培地が使用される。血液因子の組換え産生のためのこの血清含有培地の使用は言及されなかった。   Patent Document 5 describes cell culture in a medium containing a glutamine-containing protein hydrolyzate whose free amino acid content is less than 15% of the total weight of the protein and whose peptide has a molecular weight of less than 44 kD ( (See Patent Document 5). As a medium for cell culture, a minimally synthetic medium is used as a basal medium in which fetal calf serum, gentamicin and mercapto-ethanol are added in addition to the protein hydrolyzate, among others. The use of this serum-containing medium for recombinant production of blood factors was not mentioned.

特許文献６は蛋白質非含有条件下で付着性細胞の増殖を可能にする特別な合成表面につき記載している（特許文献６参照）。   Patent Document 6 describes a special synthetic surface that enables the growth of adherent cells under protein-free conditions (see Patent Document 6).

細胞増殖を刺激するために、ヒトインスリンを過剰発現するＣＨＯ細胞が、インスリンがそれに共有結合される人工的基質上で増殖された（非特許文献２参照）。   To stimulate cell proliferation, CHO cells overexpressing human insulin were grown on an artificial substrate to which insulin is covalently bound (see Non-Patent Document 2).

Ｒｅｉｔｅｒ等は、担体上の高密度の血清含有培地中で最初に増殖されたｒ−ＣＨＯ細胞の固定化および、細胞培養上澄み液中への蛋白質の連続的放出が認められる産生相中の、蛋白質非含有培地中での固定化細胞のその後の潅流培養につき記載している（非特許文献３参照）。そこで、細胞は蛋白質非含有培地中に１０世代未満維持された。   Reiter et al. Described a protein in a production phase in which immobilization of r-CHO cells initially grown in a high-density serum-containing medium on a carrier and continuous release of the protein into the cell culture supernatant were observed. It describes the subsequent perfusion culture of immobilized cells in a non-containing medium (see Non-Patent Document 3). Thus, cells were maintained in a protein-free medium for less than 10 generations.

連続細胞株、とりわけＶＥＲＯ細胞に対する、蛋白質非含有条件下の大規模細胞培養物の有効な調製の先行方法が記載されている（特許文献７参照）。そこでは細胞が最初のアンプルから工業的な大規模の１２００リットルまでの血清および蛋白質非含有条件下で増殖される。   Prior methods for the effective preparation of large-scale cell cultures under protein-free conditions for continuous cell lines, especially VERO cells, have been described (see Patent Document 7). There, cells are grown under serum and protein free conditions from the original ampoule to industrial large scale 1200 liters.

当初、血清含有条件下で増殖された細胞を蛋白質非含有培地に適応させることは通常、長時間を要する、むしろ面倒な過程であり、更に、発現蛋白質の収率および組換えＣＨＯ細胞の産生率は血清含有条件に比較して蛋白質非含有培地中への適応後に著しく低下することが繰り返し認められた（非特許文献４参照）。これは変化した培養条件による組換えクローンの不安定性もしくは増殖低下の結果である。安定な最初のクローンの使用にもかかわらず、変化された発酵条件のために大部分の細胞は繰り返して、減少した発現を伴う細胞もしくは非産生細胞になり、それが産生過程中に産生細胞を駆逐し、それにより発酵細胞の培養体は最終的に、大部分、非産生細胞もしくは低い発現を有するような細胞から成る。
米国特許第５，８９１，９９６号明細書 国際公開第９７／１９１１１号パンフレット 国際公開第９７／０５２４０号パンフレット 特開平２−６９６００１号公報 国際公開第９６／２６２６６号パンフレット 米国特許第５，３９３，６６８号明細書 国際公開第９６／１５２３１号パンフレット Ｇａｖｉｌｏｎｄｏ ｅｔ ａｌ．Ｈｙｂｒｉｄｏｍａ ９ Ｎｏ．５，１９９９ Ｉｔｏ ｅｔ ａｌ．１９９６ ＰＮＡＳ Ｕ．Ｓ．Ａ．９３：３５９８−３６０１ Ｒｅｉｔｅｒ ｅｔ ａｌ．，１９９２，Ｃｙｔｏｔｅｃｈｎｏｌｏｇｙ ９：２４７−２５３ Ｐａｔｅｒｓｏｎ ｅｔ ａｌ．１９９４．Ａｐｐｌ．Ｍｉｃｒｏｂｉｏｌ．Ｂｉｏｔｅｃｈｎｏｌ．４０：６９１−６５８ Initially, adapting cells grown under serum-containing conditions to a protein-free medium is usually a time-consuming rather rather tedious process, and also the yield of expressed protein and the production rate of recombinant CHO cells. Was repeatedly observed to be significantly reduced after adaptation to a protein-free medium as compared to serum-containing conditions (see Non-Patent Document 4). This is a result of instability or reduced growth of recombinant clones due to altered culture conditions. Despite the use of a stable initial clone, due to the altered fermentation conditions, most cells will repeatedly become cells with reduced expression or non-producing cells, which produce cells during the production process. Expelled so that the culture of fermented cells finally consists mostly of non-producing cells or cells with low expression.
US Pat. No. 5,891,996 International Publication No. 97/19111 Pamphlet International Publication No. 97/05240 Pamphlet JP-A-2-696001 International Publication No. 96/26266 Pamphlet US Pat. No. 5,393,668 International Publication No. 96/15231 Pamphlet Gavilondo et al. Hybridoma 9 No. 5,1999 Ito et al. 1996 PNAS U. S. A. 93: 3598-3601 Reiter et al. , 1992, Cytotechnology 9: 247-253. Paterson et al. 1994. Appl. Microbiol. Biotechnol. 40: 691-658

本発明において、血清および蛋白質非含有培地に適応された安定な細胞株を開発する方法が確立された。この方法に従って、幾つかのクローンが蛋白質非含有培地中で単離された。
蛋白質非含有培地への細胞株の２段階の適応
本方法は血清を補助添加されたもしくは血清非含有培地から蛋白質非含有培地への適応の直接的方法を実施することができない哺乳動物の細胞株を含んで成る。 In the present invention, a method for developing a stable cell line adapted to serum and protein-free medium has been established. According to this method, several clones were isolated in protein-free medium.
Two-step adaptation of cell lines to protein-free medium This method is a mammalian cell line that is not able to carry out the direct method of adaptation from serum-free medium to protein-free medium with serum supplementation Comprising.

本発明の方法は蛋白質非含有培地（ＰＦＭ）への細胞株の適応中の２段階の過程から成る。   The method of the invention consists of a two-step process during the adaptation of the cell line to protein-free medium (PFM).

非臨界段階と考えられる第１段階：蛋白質含量の減少が細胞生存率の喪失を伴わずに起り、蛋白質濃度の各段階における集団倍加時間の重大な減少はない。非臨界段階は通常５〜０．５ｍｇ／ｍＬの間の培地中蛋白質濃度で認められ、培養物は最初の培地中とほとんど同様な増殖速度を示す。   First stage, considered a non-critical stage: The decrease in protein content occurs without loss of cell viability, and there is no significant reduction in population doubling time at each stage of protein concentration. The non-critical stage is usually observed at protein concentrations in the medium between 5 and 0.5 mg / mL, and the culture shows a growth rate almost similar to that in the original medium.

この第１段階は８０〜１００％の間の細胞株生存率を伴って開始し、細胞は、細胞生存率が０％に低下する臨界蛋白質濃度まで、連続的な蛋白質濃度減少を伴う培地中で増殖される。この蛋白質濃度が次の段階の出発点である。   This first phase begins with a cell line viability between 80-100%, and the cells are in a medium with a continuous protein concentration reduction to a critical protein concentration at which the cell viability drops to 0%. Proliferated. This protein concentration is the starting point for the next step.

臨界段階と考えられる第２段階：この段階においては、細胞生存率および細胞の集団倍加時間の減少がおこり、蛋白質濃度の１段階からもう１つの段階に適応するために時間がよりかかるであろう。適応過程中にバイパスできない培地中の臨界蛋白質濃度が存在する。これらの臨界蛋白質濃度は各組換え細胞株に特異的であるが、通常、０．６ｍｇ／ｍＬ未満である。細胞が一旦これらの臨界蛋白質濃度で最初の生存率および増殖速度を回復した後は、より低い蛋白質濃度をもつ（ｆｏｌｌｏｗ）条件に継代培養することができる。   Second stage, considered critical stage: In this stage, cell viability and cell population doubling time will decrease, and it will take more time to adapt from one stage of protein concentration to another . There are critical protein concentrations in the medium that cannot be bypassed during the adaptation process. These critical protein concentrations are specific for each recombinant cell line, but are usually less than 0.6 mg / mL. Once cells have recovered their initial viability and growth rate at these critical protein concentrations, they can be subcultured to conditions with lower protein concentrations.

一旦臨界蛋白質濃度が固定されると、細胞の増殖を支持する閉じられた（ｃｌｏｓｅｄ）より高い蛋白質濃度が臨界前蛋白質濃度と考えられる。臨界前蛋白質濃度から出発して、それは細胞が最初の生存率および増殖速度を回復するまで緩徐に低下される。   Once the critical protein concentration is fixed, a protein concentration higher than the closed that supports cell growth is considered the pre-critical protein concentration. Starting from the pre-critical protein concentration, it is slowly lowered until the cells recover their initial viability and growth rate.

臨界段階において蛋白質濃度を減少させる段階の選択される組み合わせは、次の関係：
Ｖａｄａｐｔ．＝Δ蛋白質濃度／ΔＴａｄａｐｔ．
として計算される、この段階における総適応時間および適応速度（Ｖａｄａｐｔ．）を決定するであろう。しかし、この段階の組み合わせは臨界濃度を含む各蛋白質濃度に対する適応に要する時間に対しては影響をもたないであろう。非臨界段階の最後および臨界蛋白質濃度を決定するためには、毎回蛋白質濃度を２倍に減少することにより、所望の蛋白質非含有培地中の細胞培養物の連続的希釈による、段階的適応を実施することが必要である（表１）。この減少は血清濃度の減少もしくは異なるレベルの何か蛋白質濃度の高い血清代用物を基礎培地に補助添加することにより実施することができる。 The selected combination of steps to reduce the protein concentration in the critical step has the following relationship:
V adapt. = Δ protein concentration / ΔT adapt.
Will determine the total adaptation time and adaptation speed (V adapt. ) At this stage. However, this combination of steps will not affect the time required to adapt to each protein concentration, including the critical concentration. To determine the end of the non-critical stage and the critical protein concentration, stepwise adaptation is performed by serial dilution of the cell culture in the desired protein-free medium by doubling the protein concentration each time It is necessary to do (Table 1). This reduction can be accomplished by reducing serum concentration or supplementing the basal medium with a serum substitute with a different protein level at a different level.

Figure 2006503879
Figure 2006503879

適応法を開始する前に、細胞は通常細胞を培養するために使用される標準培地中でＴ−フラスコ中で８０％を超える生存率を伴って維持されなければならない。   Before initiating the adaptation method, the cells must be maintained with a viability in excess of 80% in the T-flask in the standard medium normally used to culture the cells.

適応法は下記の段階に従って段階毎に実施される。   The adaptation method is carried out step by step according to the following steps.

ｉ．標準細胞培地（最初の蛋白質濃度をもつ）を使用する組換え細胞株で６−ウェルの培養プレート中の３ウェルに播種する。細胞密度は１〜５×１０５細胞／ｍＬの範囲内になければならない。４８時間後に、上澄み液の半分を新鮮な、蛋白質非含有培地により置き換え、それにより出発状態の５０％の最終蛋白質濃度を与える。 i. Seed in 3 wells in a 6-well culture plate with recombinant cell line using standard cell culture medium (with initial protein concentration). The cell density should be in the range of 1-5 × 10 5 cells / mL. After 48 hours, half of the supernatant is replaced with fresh, protein-free medium, thereby giving a starting protein concentration of 50%.

ｉｉ．４８時間毎に、上澄み液を出発状態の５０％の蛋白質濃度をもつ新鮮な培地により完全に置き換える。   ii. Every 48 hours, the supernatant is completely replaced with fresh medium with a starting protein concentration of 50%.

ｉｉｉ．細胞をこの蛋白質濃度下で集密状態まで増殖させる。   iii. Cells are grown to confluence under this protein concentration.

ｉｖ．段階ｉｉｉ．からの細胞を出発状態の５０％の蛋白質濃度をもつ培地中１〜５×１０５細胞／ｍＬの範囲内の密度で少なくとも３ウェルに播種する。４８時間後に、上澄み液の半分を新鮮な、蛋白質非含有培地により置き換え、それにより前の状態の５０％の最終蛋白質濃度を与える。 iv. Stage iii. Are seeded in at least 3 wells at a density in the range of 1-5 × 10 5 cells / mL in medium with a starting 50% protein concentration. After 48 hours, half of the supernatant is replaced with fresh, protein-free medium, thereby giving a final protein concentration of 50% of the previous state.

ｖ．４８時間毎に、上澄み液を前の状態の５０％の蛋白質濃度をもつ新鮮な培地により完全に置き換える。   v. Every 48 hours, the supernatant is completely replaced with fresh medium with a protein concentration of 50% of the previous state.

ｖｉ．．細胞をこの蛋白質濃度下で集密状態まで増殖させる。   vi. . Cells are grown to confluence under this protein concentration.

ｖｉｉ．（ｉｖ）〜（ｖｉ）の段階を繰り返し、各サイクル中蛋白質濃度を前のサイクルの濃度の５０％まで減少させる。細胞死をもたらす蛋白質濃度に到達するまでこの方法を繰り返す。   vii. Repeat steps (iv)-(vi) to reduce the protein concentration during each cycle to 50% of the concentration of the previous cycle. This method is repeated until a protein concentration that results in cell death is reached.

ｖｉｉｉ．２〜６×１０５細胞／ｍＬの範囲内の密度で、少なくとも３ウェル中に、臨界前蛋白質濃度中に増殖する、８０％以上の生存率を伴う細胞培養物からの細胞を播種する。細胞は臨界前蛋白質濃度中で増殖し、４８時間後に上澄み液の２５％を新鮮な、蛋白質非含有培地により置き換え、これによりそれを臨界前蛋白質濃度の７５％の最終蛋白質濃度にさせる。 viii. Seed cells from a cell culture with a viability of 80% or higher, growing in a pre-critical protein concentration, in at least 3 wells at a density in the range of 2-6 × 10 5 cells / mL. The cells grow in the pre-critical protein concentration and after 48 hours, 25% of the supernatant is replaced with fresh, protein-free medium, which brings it to a final protein concentration of 75% of the pre-critical protein concentration.

ｉｘ．４８時間毎に上澄み液を臨界前蛋白質濃度の７５％の蛋白質濃度をもつ新鮮な培地により完全に置き換える。   ix. Every 48 hours, the supernatant is completely replaced with fresh medium with a protein concentration of 75% of the precritical protein concentration.

ｘ．細胞をこの蛋白質濃度下で集密状態まで増殖させる。
ｘｉ．段階（ｘ）からの細胞を臨界前蛋白質濃度の７５％の蛋白質濃度をもつ培地中に２〜６×１０５細胞／ｍＬの範囲内の密度で少なくとも３ウェル中に播種する。４８時間後に上澄み液の２５％を新鮮な、蛋白質非含有培地により置き換え、これによりそれを前段階の濃度の７５％の最終蛋白質濃度にさせる。 x. Cells are grown to confluence under this protein concentration.
xi. Cells from step (x) are seeded in at least 3 wells at a density in the range of 2-6 × 10 5 cells / mL in a medium having a protein concentration of 75% of the precritical protein concentration. After 48 hours, 25% of the supernatant is replaced with fresh, protein-free medium, which brings it to a final protein concentration of 75% of the previous concentration.

ｘｉｉ．４８時間毎に上澄み液を段階（ｘ）の濃度の７５％の蛋白質濃度をもつ新鮮な培地により完全に置き換える。   xii. Every 48 hours the supernatant is completely replaced with fresh medium with a protein concentration of 75% of the concentration of step (x).

ｘｉｉｉ．細胞をこの蛋白質濃度下で集密状態まで増殖させる。
ｘｉｖ．（ｘｉ）〜（ｘｉｉｉ）の段階を繰り返し、各サイクル中、蛋白質濃度を前のサイクルの濃度の７５％まで減少させ、次にこの方法を、細胞生存率のあらゆる喪失および集団倍加時間の減少を引き起こさない蛋白質濃度に到達するまで繰り返す。細胞をより低い蛋白質濃度をもつ培地に移し、それらは細胞生存率のあらゆる喪失および第１継代培養前の集団培養時間の減少を伴わずに増殖することができる時に、細胞は再度、非臨界段階に到達したと考えられ、蛋白質非含有培地（０ｍｇ／ｍＬの蛋白質濃度）中にそれを直接播種する。 xiii. Cells are grown to confluence under this protein concentration.
xiv. Repeat steps (xi)-(xiii) and during each cycle reduce the protein concentration to 75% of the concentration of the previous cycle, then use this method to reduce any loss of cell viability and population doubling time. Repeat until a protein concentration that does not cause it is reached. When the cells are transferred to a medium with a lower protein concentration and they can grow without any loss of cell viability and a decrease in population culture time prior to the first passage, the cells are again non-critical. The stage is considered reached and is seeded directly in protein-free medium (0 mg / mL protein concentration).

本発明の方法において、最初の培地は５〜１０％の間の範囲の胎児コウシ血清を含有する。   In the method of the present invention, the initial medium contains fetal calf serum in the range between 5-10%.

蛋白質非含有培地中での増殖に適応された哺乳動物の細胞株は骨髄腫、特にＮＳＯ細胞である。   Mammalian cell lines adapted for growth in protein-free medium are myeloma, especially NSO cells.

本発明はまた、ポリペプチドもしくは組換え蛋白質でトランスフェクトされたＮＳＯ細胞株を使用する時、特にそれらが組換え抗体もしくはその断片をエンコードする配列でトランスフェクトされる時に有用であることができると考えられる。蛋白質非含有培地中で増殖する本発明の方法により修飾される哺乳動物の細胞株も開示される。   The invention can also be useful when using NS0 cell lines transfected with a polypeptide or recombinant protein, particularly when they are transfected with a sequence encoding a recombinant antibody or fragment thereof. Conceivable. Also disclosed are mammalian cell lines that are modified by the methods of the invention to grow in protein-free media.

本発明の異なる態様において、蛋白質非含有培地中で増殖する抗−ＥＧＦ受容体ｈＲ３、抗−ＣＤ６ Ｔ１ｈＴ、抗−ＣＤ３ Ｔ３Ｑ抗体もしくはそれらの断片から成る群から選択されるヒト化もしくはキメラ抗体を発現するあらゆる哺乳動物の細胞株、従ってこの細胞株により分泌される抗体が開示される。   In a different embodiment of the invention, expressing a humanized or chimeric antibody selected from the group consisting of anti-EGF receptor hR3, anti-CD6 T1hT, anti-CD3 T3Q antibody or fragments thereof growing in protein-free medium Any mammalian cell line, and thus antibodies secreted by this cell line, is disclosed.

本発明の方法により得られる細胞株は少なくとも４０世代に対して蛋白質非含有培地中で安定に増殖する。   The cell line obtained by the method of the present invention stably grows in a protein-free medium for at least 40 generations.

蛋白質非含有培地への組換え細胞株ｈＲ３の適応。
ヒト化抗−ＥＧＦヒト受容体ｈＲ３モノクロナール抗体の軽鎖および重鎖を発現するためのベクトル構造をもつ骨髄腫ＮＳＯ細胞株のトランスフェクションにより、組換え細胞株ｈＲ３を得た。この細胞株の蛋白質非含有培地への適応は培地の蛋白質含量の２段階の減少により前記の方法に従って実施した。 Adaptation of recombinant cell line hR3 to protein-free medium.
Recombinant cell line hR3 was obtained by transfection of a myeloma NSO cell line with a vector structure to express the light and heavy chains of the humanized anti-EGF human receptor hR3 monoclonal antibody. Adaptation of this cell line to a protein-free medium was performed according to the method described above by a two-step decrease in the protein content of the medium.

これらの細胞を１０％のＦＢＳを補助添加されたＲＰＭＩ−１６４０培地中で培養した。蛋白質濃度が０．１５ｍｇ／ｍＬである時にＲＰＭＩ−１６４０蛋白質非含有培地に蛋白質到達補助添加剤のＮｕｔｒｉｄｏｍａ ＮＳ（Ｂｏｈｅｒｉｎｇｅｒ Ｍａｎｈｅｉｎｎ）を添加することによりＦＢＳを置き換えた。最初の培地中の蛋白質含量の減少をＲＦＨＭ−ＩＩ蛋白質非含有培地（Ｇｉｂｃｏ）による連続希釈により実施した。   These cells were cultured in RPMI-1640 medium supplemented with 10% FBS. When the protein concentration was 0.15 mg / mL, FBS was replaced by adding Nutridoma NS (Boheringer Manheimn), a protein reaching auxiliary additive, to the RPMI-1640 protein-free medium. Reduction of the protein content in the initial medium was performed by serial dilution with RFHM-II protein-free medium (Gibco).

図１および２はそれぞれ、臨界濃度および適応速度Ｖａｄａｐｔ．の計算の結果を示す。 1 and 2 show the critical concentration and the adaptation rate V adapt. The result of calculation is shown.

組換え最初株Ｔ１ｈＴの蛋白質非含有培地への適応。
組換え細胞株Ｔ１ｈＴをエピトープＴ抑制法によりヒト化された抗−ヒトＣＤ６モノクロナール抗体の軽鎖および重鎖を発現するためのベクトル構造をもつ骨髄腫ＮＳＯ細胞株のトランスフェクションにより得た。 Adaptation of recombinant first strain T1hT to protein-free medium.
Recombinant cell line T1hT was obtained by transfection of a myeloma NSO cell line with a vector structure to express the light and heavy chains of an anti-human CD6 monoclonal antibody that was humanized by epitope T inhibition.

蛋白質／非含有培地へのこの細胞株の適応を培地の蛋白質含量の２段階減少によりポイント２に記載された方法に従って実施した。   Adaptation of this cell line to protein / free medium was performed according to the method described in point 2 by a two-step decrease in the protein content of the medium.

これらの細胞は最初に１０％のＦＢＳを補助添加されたＲＰＭＩ−１６４０培地中で培養した。蛋白質含量の減少はＧｉｂｃｏからのＰＦＨＭ−ＩＩ蛋白質非含有培地による最初の培地の連続的希釈により実施した。   These cells were initially cultured in RPMI-1640 medium supplemented with 10% FBS. Reduction of protein content was performed by serial dilution of the initial medium with PFHM-II protein-free medium from Gibco.

臨界濃度および適応速度Ｖａｄａｐｔ．の計算結果はそれぞれ、図３および４に示す。 Critical concentration and adaptation speed V adapt. The calculation results are shown in FIGS. 3 and 4, respectively.

組換え細胞株Ｔ３Ｑの蛋白質非含有培地への適応。
組換え細胞株Ｔ３Ｑを、ヒトリンパ球上のＣＤ３受容体を認識するヒト化モノクロナール抗体Ｔ３Ｑの軽鎖および重鎖を発現するためのベクトル構造をもつ骨髄腫ＮＳＯ細胞株のトランスフェクションにより得た。 Adaptation of recombinant cell line T3Q to protein-free medium.
Recombinant cell line T3Q was obtained by transfection of a myeloma NSO cell line with a vector structure to express the light and heavy chains of humanized monoclonal antibody T3Q that recognizes the CD3 receptor on human lymphocytes.

蛋白質／非含有培地へのこの細胞株の適応を培地の蛋白質含量の２段階減少によりポイント２に記載された方法に従って実施した。   Adaptation of this cell line to protein / free medium was performed according to the method described in point 2 by a two-step decrease in the protein content of the medium.

これらの細胞を最初に１０％のＦＢＳを補助添加されたＲＰＭＩ−１６４０培地中で培養した。蛋白質含量の減少をＧｉｂｃｏからのＲＦＨＭ−ＩＩ蛋白質非含有培地中への連続希釈により実施した。   These cells were initially cultured in RPMI-1640 medium supplemented with 10% FBS. Reduction of protein content was performed by serial dilutions into Gibco's RFHM-II protein-free medium.

臨界濃度および適応速度Ｖａｄａｐｔ．の計算の結果を図５および６それぞれに示す。 Critical concentration and adaptation speed V adapt. The calculation results are shown in FIGS. 5 and 6, respectively.

蛋白質濃度の逆数の自然対数との、各蛋白質濃度にｈＲ３細胞を適応するために要する（生存率および倍加時間の回復までの）時間の関係。ｈ−Ｒ３細胞株の臨界濃度値：−０．３２および０．１１ｍｇ／ｍＬの総蛋白質濃度。The relationship between the natural logarithm of the reciprocal of protein concentration and the time required to adapt hR3 cells to each protein concentration (until recovery of viability and doubling time). Critical concentration values for h-R3 cell line: -0.32 and 0.11 mg / mL total protein concentration. ｈ−Ｒ３細胞株の蛋白質濃度の逆数の自然対数との、適応法の開始からの総合時間の関係。臨界段階中のｈ−Ｒ３細胞株の適応速度の値−０．００５３ｍｇ／日。The relationship of the total time from the start of the adaptation method to the natural logarithm of the reciprocal of the protein concentration of the h-R3 cell line. Adaptation rate value of the h-R3 cell line during the critical phase-0.0053 mg / day. 蛋白質濃度の逆数の自然対数との、各蛋白質濃度にＴ１ｈＴ細胞を適応させるために要する（生存率および倍加時間を回復するまでの）時間の関係。Ｔ１ｈＴ細胞株の臨界濃度の値−０．１２および０．０１ｍｇ／ｍＬの総蛋白質濃度。Relationship between the natural logarithm of the reciprocal of protein concentration and the time required to adapt T1hT cells to each protein concentration (until recovery of viability and doubling time). Values for critical concentration of T1hT cell line -total protein concentration of 0.12 and 0.01 mg / mL. Ｔ１ｈＴ細胞株の蛋白質濃度の逆数の自然対数との、適応法の開始からの総時間の関係。Ｔ１ｈＴ細胞株の適応速度の値−０．００１４ｍｇ／日。The relationship of the total time from the start of the adaptation method to the natural logarithm of the reciprocal of the protein concentration of the T1hT cell line. Adaptation rate value of T1hT cell line-0.0014 mg / day. 蛋白質濃度の逆数の自然対数との、各蛋白質濃度にＴ３Ｑ細胞を適応させるために要する（生存率および倍加時間を回復するまでの）時間の関係。Ｔ３Ｑ細胞株の臨界濃度の値−０．６３ｍｇ／ｍＬの総蛋白質濃度。Relationship between the natural logarithm of the reciprocal of protein concentration and the time required to adapt T3Q cells to each protein concentration (until recovery of viability and doubling time). T3Q cell line critical concentration value-0.63 mg / mL total protein concentration. Ｔ３Ｑ細胞株の蛋白質濃度の逆数の自然対数との、適応法の開始からの総時間の関係。Ｔ３Ｑ細胞株の適応速度の値−０．００１７２ｍｇ／日。The relationship of the total time from the start of the adaptation method to the natural logarithm of the reciprocal of the protein concentration of the T3Q cell line. Adaptation rate value for the T3Q cell line-0.00172 mg / day.

Claims (20)

２段階：
Ｉ．細胞株生存率が８０〜１００％の間であり、細胞生存率が０％に低下する臨界蛋白質濃度まで連続的蛋白質濃度減少を伴う培地中で細胞が増殖される第１段階、
ＩＩ．一旦臨界濃度が前以て決定された後、次に細胞増殖が可能で、かつそれが細胞培養物まで蛋白質濃度を緩徐に減少させる開始地点であるような蛋白質濃度が最初の細胞生存率および集団倍加時間に到達するように、臨界前濃度が固定される第２段階、
を含んで成る、血清および蛋白質非含有培地中での増殖に適応された哺乳動物の細胞株の取得方法。 Two steps:
I. A first stage in which the cells are grown in a medium with a continuous protein concentration reduction to a critical protein concentration where the cell line viability is between 80-100% and the cell viability is reduced to 0%;
II. Once the critical concentration has been determined in advance, the protein concentration is such that the next cell growth is possible and that is the starting point for a gradual decrease in protein concentration to the cell culture. A second stage in which the pre-critical concentration is fixed to reach the doubling time;
A method of obtaining a mammalian cell line adapted for growth in a serum- and protein-free medium comprising 第１段階が以下の段階：
ｉ．標準細胞培地（最初の蛋白質濃度をもつ）を使用する組換え細胞株で６ウェルの培養プレート中の３ウェルに播種する。細胞密度は１〜５×１０５細胞／ｍＬの範囲内になければならない。４８時間後に、上澄み液の半分を新鮮な、蛋白質非含有培地により置き換え、それにより出発状態の５０％の最終蛋白質濃度を与える。
ｉｉ．４８時間毎に、上澄み液を出発状態の５０％の蛋白質濃度をもつ新鮮な培地により完全に置き換える。
ｉｉｉ．細胞をこの蛋白質濃度下で集密状態まで増殖させる。
ｉｖ．段階ｉｉｉ．からの細胞を出発状態の５０％の蛋白質濃度をもつ培地中１〜５×１０５細胞／ｍＬの範囲内の密度で少なくとも３ウェルに播種する。４８時間後に、上澄み液の半分を新鮮な、蛋白質非含有培地により置き換え、それにより前の状態の５０％の最終蛋白質濃度を与える。
ｖ．４８時間毎に、上澄み液を前の状態の５０％の蛋白質濃度をもつ新鮮な培地により完全に置き換える。
ｖｉ．細胞をこの蛋白質濃度下で集密状態まで増殖させる。
ｖｉｉ．（ｉｖ）〜（ｖｉ）の段階を繰り返し、各サイクル中に、前のサイクルの濃度の５０％まで蛋白質濃度を減少させる。細胞死をもたらす蛋白質濃度に到達するまでこの方法を繰り返す。
により構成される請求項１記載の方法。 The first stage is as follows:
i. Seed in 3 wells in a 6 well culture plate with recombinant cell line using standard cell culture medium (with initial protein concentration). The cell density should be in the range of 1-5 × 10 5 cells / mL. After 48 hours, half of the supernatant is replaced with fresh, protein-free medium, thereby giving a starting protein concentration of 50%.
ii. Every 48 hours, the supernatant is completely replaced with fresh medium with a starting protein concentration of 50%.
iii. Cells are grown to confluence under this protein concentration.
iv. Stage iii. Are seeded in at least 3 wells at a density in the range of 1-5 × 10 5 cells / mL in medium with a starting 50% protein concentration. After 48 hours, half of the supernatant is replaced with fresh, protein-free medium, thereby giving a final protein concentration of 50% of the previous state.
v. Every 48 hours, the supernatant is completely replaced with fresh medium with a protein concentration of 50% of the previous state.
vi. Cells are grown to confluence under this protein concentration.
vii. Steps (iv)-(vi) are repeated and the protein concentration is reduced to 50% of the concentration of the previous cycle during each cycle. This method is repeated until a protein concentration that results in cell death is reached.
The method of claim 1 comprising: 第２段階が以下の段階：
ｖｉｉｉ．２〜６×１０５細胞／ｍＬの範囲内の密度で少なくとも３ウェル中に臨界前蛋白質濃度中に増殖する、８０％以上の生存率を伴う細胞培養物からの細胞を播種する。細胞は臨界前蛋白質濃度中で増殖し、４８時間後に上澄み液の２５％を新鮮な、蛋白質非含有培地により置き換え、これによりそれを臨界前蛋白質濃度の７５％の最終蛋白質濃度にさせる。
ｉｘ．４８時間毎に上澄み液を臨界前蛋白質濃度の７５％の蛋白質濃度をもつ新鮮な培地により完全に置き換える。
ｘ．細胞をこの蛋白質濃度下で集密状態まで増殖させる。
ｘｉ．段階（ｘ）からの細胞を臨界前蛋白質濃度の７５％の蛋白質濃度をもつ培地中に２〜６×１０５細胞／ｍＬの範囲内の密度で少なくとも３ウェル中に播種する。４８時間後に上澄み液の２５％を新鮮な、蛋白質非含有培地により置き換え、これによりそれを前段階の濃度の７５％の最終蛋白質濃度にさせる。
ｘｉｉ．４８時間毎に上澄み液を段階（ｘ）の濃度の７５％の蛋白質濃度をもつ新鮮な培地により完全に置き換える。
ｘｉｉｉ．細胞をこの蛋白質濃度下で集密状態まで増殖させる。
ｘｉｖ．（ｘｉ）〜（ｘｉｉｉ）の段階を繰り返し、各サイクル中、蛋白質濃度を前のサイクルの濃度の７５％まで減少させ、次にこの方法を、細胞生存率のあらゆる喪失および集団倍加時間の減少を引き起こさない蛋白質濃度に到達するまで繰り返す。細胞をより低い蛋白質濃度をもつ培地に移し、それらは細胞生存率のあらゆる喪失および第１継代培養の前の集合培養時間の減少を伴わずに増殖することができる時に、細胞は再度、非臨界段階に到達したと考えられ、蛋白質非含有培地（０ｍｇ／ｍＬの蛋白質濃度）中に直接播種する。
により構成される、請求項１記載の方法。 The second stage is:
viii. Seed cells from a cell culture with a viability of 80% or higher, growing in a pre-critical protein concentration in at least 3 wells at a density in the range of 2-6 × 10 5 cells / mL. The cells grow in the pre-critical protein concentration and after 48 hours, 25% of the supernatant is replaced with fresh, protein-free medium, which brings it to a final protein concentration of 75% of the pre-critical protein concentration.
ix. Every 48 hours, the supernatant is completely replaced with fresh medium with a protein concentration of 75% of the precritical protein concentration.
x. Cells are grown to confluence under this protein concentration.
xi. Cells from step (x) are seeded in at least 3 wells at a density in the range of 2-6 × 10 5 cells / mL in a medium having a protein concentration of 75% of the precritical protein concentration. After 48 hours, 25% of the supernatant is replaced with fresh, protein-free medium, which brings it to a final protein concentration of 75% of the previous concentration.
xii. Every 48 hours the supernatant is completely replaced with fresh medium with a protein concentration of 75% of the concentration of step (x).
xiii. Cells are grown to confluence under this protein concentration.
xiv. Repeat steps (xi)-(xiii) and during each cycle reduce the protein concentration to 75% of the concentration of the previous cycle, then use this method to reduce any loss of cell viability and population doubling time. Repeat until a protein concentration that does not cause it is reached. When the cells are transferred to a medium with a lower protein concentration and they can grow without any loss of cell viability and a decrease in the assembly time before the first passage, the cells are again non- It is considered that the critical stage has been reached, and seeded directly in protein-free medium (protein concentration of 0 mg / mL).
The method of claim 1, comprising: 細胞が最初に播種される血清および蛋白質含有培地が５％〜１０％の間の胎児ウシ血清を含んで成る請求項１〜３に記載の方法。   4. The method according to claims 1 to 3, wherein the serum and the protein-containing medium on which the cells are initially seeded comprise between 5% and 10% fetal bovine serum. 血清および蛋白質非含有培地中での増殖に適応された哺乳動物の細胞株が骨髄腫である請求項１〜４に記載の方法。   The method according to claims 1 to 4, wherein the mammalian cell line adapted for growth in serum- and protein-free medium is myeloma. 骨髄腫がＮＳＯ細胞株である請求項５記載の方法。   6. The method of claim 5, wherein the myeloma is an NSO cell line. 前記ＮＳＯ細胞株が組換えポリペプチドもしくは組換え蛋白質をエンコードする配列を含む請求項６記載の方法。   7. The method of claim 6, wherein said NSO cell line comprises a sequence encoding a recombinant polypeptide or protein. 組換えポリペプチドもしくは組換え蛋白質をエンコードする配列が組換え抗体もしくはその断片をコード化する請求項７記載の方法。   8. The method of claim 7, wherein the sequence encoding the recombinant polypeptide or protein encodes a recombinant antibody or fragment thereof. 前記細胞株が血清および蛋白質非含有培地中での増殖に適応されている、請求項１〜８の方法により得られる哺乳動物の細胞株。   A mammalian cell line obtained by the method of claims 1-8, wherein the cell line is adapted for growth in serum and protein-free medium. 前記細胞株が骨髄腫である請求項９記載の哺乳動物の細胞株。   The mammalian cell line according to claim 9, wherein the cell line is myeloma. 前記細胞株がＮＳＯ細胞株である請求項１０記載の哺乳動物の細胞株。   The mammalian cell line according to claim 10, wherein the cell line is an NSO cell line. ＮＳＯ細胞株が組換えポリペプチドもしくは組換え蛋白質をエンコードする配列を含む請求項１１記載の哺乳動物の細胞株。   12. The mammalian cell line of claim 11, wherein the NSO cell line comprises a sequence encoding a recombinant polypeptide or protein. 組換えポリペプチドもしくは組換え蛋白質をエンコードする配列が組換え抗体もしくはその断片をコード化する請求項１２記載の哺乳動物細胞株。   The mammalian cell line of claim 12, wherein the sequence encoding the recombinant polypeptide or protein encodes a recombinant antibody or fragment thereof. 配列がヒト化組換え抗体抗−ＥＧＦ−Ｒ ｈＲ３もしくはその断片をコード化する請求項１３記載の哺乳動物細胞株。   14. The mammalian cell line of claim 13, wherein the sequence encodes a humanized recombinant antibody anti-EGF-R hR3 or a fragment thereof. 配列がヒト化組換え抗−ＣＤ６抗体Ｔ１ｈＴもしくはその断片をコード化する請求項１３記載の哺乳動物細胞株。   14. The mammalian cell line of claim 13, wherein the sequence encodes a humanized recombinant anti-CD6 antibody T1hT or a fragment thereof. 配列がキメラ組換え抗−ＣＤ３抗体Ｔ３Ｑもしくはその断片をコード化する請求項１３記載の哺乳動物細胞株。   14. The mammalian cell line of claim 13, wherein the sequence encodes a chimeric recombinant anti-CD3 antibody T3Q or a fragment thereof. 血清および蛋白質非含有培地中での増殖に適応された哺乳動物細胞株を取得するための請求項１〜８記載の方法の使用。   Use of the method according to claims 1 to 8 for obtaining mammalian cell lines adapted for growth in serum and protein-free medium. 請求項１〜８の方法により得られる細胞株により分泌されるＥＧＦ−Ｒ ｈＲ３もしくはその断片に対するヒト化モノクロナール抗体。   A humanized monoclonal antibody against EGF-R hR3 or a fragment thereof secreted by a cell line obtained by the method of claims 1-8. 請求項１〜８の方法により得られる細胞株により分泌されるＣＤ６抗原Ｔ１ｈＴもしくはその断片に対するヒト化モノクロナール抗体。   A humanized monoclonal antibody against CD6 antigen T1hT or a fragment thereof secreted by a cell line obtained by the method of claims 1-8. 請求項１〜８の方法により得られる細胞株により分泌されるＣＤ３抗原Ｔ３Ｑもしくはその断片に対するキメラモノクロナール抗体。   A chimeric monoclonal antibody against CD3 antigen T3Q or a fragment thereof secreted by a cell line obtained by the method of claims 1-8.
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JPN7009003896, Ｍｏｌ． Ｂｉｏｔｅｃｈｎｏｌ．， ｖｏｌ． １５， ｐａｇｅｓ ２４９−２５７ （２０００） *

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LV13361B (en) 2006-02-20
CN1714147A (en) 2005-12-28
AU2003273727B2 (en) 2008-04-03
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